This investigation will explore the role of antibodies (Ab) in Dengue virus (DENV) pathogenesis using a two- part hypothesis-driven approach to studying antibody-dependent enhancement (ADE) of DENV infection and elucidating the immunopathological mechanisms responsible for vascular leakage in vitro and in vivo. Part one examines the ability of human monoclonal antibodies (HMAb) to enhance endothelial cell (EC) infectivity in vitro. We hypothesize that DENV-specific Abs can enhance infection of ECs leading to increased loss of EC barrier function and changes in vascular permeability. HMAbs generated from previously DENV-infected patients that exhibit ADE in vitro will be used to determine the ability of Abs to enhance DENV infection of ECs, resulting in disruption of vascular integrity in vitro. The second component involves the ability of HMAbs to cause ADE of DENV infection in vivo using the ferret as a novel small animal model of DENV infection. Our hypothesis is that in vitro EC infection correlates with in vivo pathology seen in DENV infection, specifically that the presence of Ab enhances EC infectivity and induces histopathological changes in the microvasculature resulting in vascular leak. Ferrets that are either mock treated or treated with HMAbs will subsequently be challenged with DENV and assessed for signs of enhanced infection, including clinical manifestations, increased viremia, and signs of vascular leakage. Results of these studies will determine the precise role of Abs in DENV pathogenesis, specifically their ability to induce vascular leak in vitro and in vivo, and will provide a clearer understanding of the role of DENV Abs in the induction of Dengue Hemorrhagic Fever/Dengue Shock Syndrome (DHF/DSS). PUBLIC HEALTH RELEVANCE: Over the past fifty years, the incidence of Dengue fever (DF), caused by Dengue virus (DENV), and Dengue Hemorrhagic Fever/Dengue Shock Syndrome (DHF/DSS), a potentially fatal complication of DF and a major cause of morbidity and mortality in children throughout the world, has increased 30-fold and has expanded into new parts of the world, including the United States, resulting in volatile outbreaks and making DENV a global public health problem. There are currently no treatments or vaccines available against DENV, and a limited understanding of the immune response to the virus coupled with the lack of suitable animal models to study the disease hinder the development of DENV antivirals and vaccines. The proposed research will study the human antibody response to DENV and utilize a new small animal model of DENV infection to gain a more complete understanding of the immune response to DENV and assess the ability to use antibodies for treatment or as tools to help develop vaccines.